roiect Objectives: The S. cerevisiae mating pheromone response pathway is arguably the best understood multi-tiered MARK signaling cascade in any eukaryote. It is now appreciated, however, that coordinating the changes in gene expression and cell morphology necessary for mating involves an elaborate network of nterlocking events, rather than a simple linear pathway. In addition, feedback mechanisms exist to modu- ate the efficiency and duration of these signaling events at essentially every step. Moreover, this signaling oathway must evoke an appropriate response upon the correct stimulus, yet avoid adventitious activation. Furthermore, it is now established that some components required for pheromone response are also utilized both for a different developmental outcome, termed filamentous/invasive growth (triggered by nutrient limita- tion) and for response to hyperosmotic stress. How different extracellular signals impinge on the same MARK elements, yet are deciphered differently, is not fully understood in any organism. For all of these reasons, yeast continues to provide opportunities to examine basic properties of the organization, specificity, Fidelity, regulation and function of MARK signaling pathways, including how certain molecules that participate in signaling can be coupled to different upstream inputs and downstream responses in the same cell type. Specific aims include: (1) Further characterization of two pivotal scaffold proteins, Ste5 and SteSO, including crystal structure determination and, in the case of Ste5, the mechanisms by which its RING-H2 domain contributes to oligomerization, intra- and intermolecular ubiquitinylation, interaction with Gpy (Ste4-Ste18) and other aspects of its function. (2) Further analysis of the function of plasma membrane phosphoinositides in the membrane recruitment of Ste5 (via its PH domain) and the Bem1 adaptor protein (via its PX domain). (3) Further development of a novel FRET-based reporter we devised recently to visualize spatiotemporal features of MARK activation in real time in live cells. (4) Examination of the biochemical basis by which pheromone signaling affects normal septin filament assembly and interdicts cytokinesis. (5) Investigation of how the MARK, Kss1, contributes to control of the apparent IRES elements in the transcripts for gene products required for invasive growth. (6) Determination at the molecular level of the mechanism by which the stress-activated MARK, Hog1, blocks inappropriate activation of the other two pathways that utilize the sameMAPKKK(Ste11). Public Health Relevance: The growth of many human tumor cells can be traced to mutations that lead directly to inappropriate and persistent MARK activation. Thus, studying fundamental aspects of MARK signaling may provide insights for the development of more effective anti-cancer therapies to ameliorate certain prevalent malignancies in people.